1. Field of the Invention
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the .sup.32 P labelling of DNA with T4 kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
Commonly used methods for detecting specific nucleic acid sequences generally involve immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labelled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The presence of hybridized material on the support is detected by autoradiography or by spectrometric methods.
Since these methods are slow and labor intensive, and generally not suitable for very low concentrations, it is desirable to develop methods with increased sensitivity and simplicity. Preferably, new methods should avoid the hazards of radioactivity and employ homogeneous assay techniques, which offer opportunities for speed and simplicity.
Recently, a method for the enzymatic amplification of specific double stranded sequences of DNA known as the polymerase chain reaction (PCR) has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the desired sequence flanked by the primers. The two different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
Another method that has recently been described is an amplification of a single stranded polynucleotide using a single polynucleotide primer. The single stranded polynucleotide that is to be amplified contains two non-contiguous sequences that are complementary to one another and, thus, are capable of hybridizing together to form a stem-loop structure. This single stranded polynucleotide may be already part of a polynucleotide analyte or may be created as the result of the presence of a polynucleotide.
One aspect of single primer amplification as it is currently described is that, if a single strand of the analyte is not capable of forming a stem-loop structure, then a single stranded polynucleotide having such a stem-loop structure must be created. This created single stranded polynucleotide must also be related to the presence of the polynucleotide analyte. The present invention allows for the advantages of single primer amplification, namely one primer and one polymerase, without the need for this added step of creating a single stranded polynucleotide with a stem-loop structure.
2. Description of the Related Art.
A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region is described by Parks, et al., Nucleic Acids Research (1991) 19(No.25): 7155-7160. Wang, et al., DNA and Cell Biology (1991) 10(No.10): 771-777 discuss the single primer-mediated polymerase chain reaction: application in cloning of two different 5'-untranslated sequences of acidic fibroblast growth factor mRNA.
Paabo, et al., discuss jumping between templates during enzymatic amplification promoted by DNA damage (J. Biol. Chem. (1990) 265(No.8): 4718-4721).
U.S. patent application Ser. Nos. 07/299,282 and 07/399,795 filed Jan. 19, 1989, and Aug. 29, 1989, respectively, describe nucleic acid amplification using a single polynucleotide primer. U.S. patent application Ser. No. 07/555,323 filed Jul. 19, 1990, discloses methods for producing a polynucleotide for use in single primer amplification. U.S. patent application Ser. No. 07/555,968 filed Jul. 19, 1990, describes a method for producing a molecule containing an intramolecular base-pair structure. U.S. patent application Ser. No. 07/776,538 filed Oct. 11, 1991, discloses methods for producing a polynucleotide for use in single primer amplification. U.S. patent application Ser. No. 07/923,079 filed Jul. 31, 1992, describes a method for introducing sequences at the 3' end of polynucleotides. The disclosures of these six applications are incorporated herein by reference in their entirety.
A process for amplifying, detecting and/or cloning nucleic acid sequences otherwise referred to as PCR is disclosed in U.S. Pat. Nos. 5,008,182, 4,965,188, 4,800,159, 4,683,195 and U.S. Pat. No. 4,683,202. Sequence polymerization by PCR is described by Saiki, et al., (1986) Science, 230: 1350-1354.
Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer is discussed by Frohman, et al., in Proc. Natl. Acad. Sci. USA (1988) 85:8998-9002. A discussion of the generation of long terminal repeats in proviral DNA by reverse transcriptase is found in "Molecular Biology of the Gene," Fourth Edition, The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif., pages 939-941. The effect of intron mutations on the splicing of Saccharomyces cerevisiae SUP53 precursor tRNA is discussed by Strobel, et al., in Molecular and Cellular Biology (1986) 6(No.7): 2674-2683. Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers is described in U.S. Pat. No. 5,043,272.
A single stranded self-hybridizing nucleic acid probe capable of repeatedly hybridizing to itself or other nucleic acids to form an amplified entity is described in U.S. patent application Ser. No. 888,058, filed Jul. 22, 1986 (Department of Health and Human Services). Methods of generating single stranded DNA by PCR are disclosed in U.S. Pat. No. 5,066,584. A method of making an oligonucleotide is described in European Patent Application No. 0194545 A2. Belgian Patent Application No. BE 904402 discloses a mold for making DNA detection probes. Gene amplification in eukaryotic cells is disclosed in U.S. Pat. No. 4,656,134.
Langer, et al., Proc. Natl. Acad. Sci. USA, (1981) 78: 6633-6637 discloses the enzymatic synthesis of biotin labelled polynucleotides and the use of these materials as novel nucleic acid affinity probes. The detection of viral genomes in cultured cells and paraffin imbedded tissue sections using biotin labelled hybridization probes is discussed by Brigati, et al., Virology, (1983) 126: 32-50. U.S. Pat. No. 4,486,539 discloses the detection of microbial nucleic acids by a one step sandwich hybridization test. Sensitive tests for malignancies based on DNA detection is described in U.S. Pat. No. 4,490,472. U.S. Pat. No. 4,480,040 discloses the sensitive and rapid diagnosis of plant viroid diseases and viruses employing radioactively labelled DNA that is complementary to the viroid or to the nucleic acid of the virus being diagnosed. European Patent Application No. 106 112 (Priority U.S. patent application 391,440 filed Jun. 23, 1982) teaches modified labelled nucleotides and polynucleotides and methods of preparing, utilizing, and detecting the same. Methods and compositions for the detection and determination of cellular DNA are disclosed in U.S. Pat. No. 4,423,153. Specific DNA probes in diagnostic microbiology are discussed in U.S. Pat. No. 4,358,535. A method for detection of polymorphic restriction sites and nucleic acid sequences is discussed in European Patent Application No. 0164054 A1. U.S. Pat. No. 4,663,283 describes a method of altering double-stranded DNA.
Genomic amplification with transcript sequencing is discussed by Stoflet, et al., Science (1988) 239:491. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al., Science (1988) 239:487. U.S. Pat. No. 4,724,202 discloses the use of non-hybridizable nucleic acids for the detection of nucleic acid hybridization. Bugawan, et al., Bio/Technology, (1988) 6:943-947 describe the use of non-radioactive oligonucleotide probes to analyze enzymatically amplified DNA for prenatal diagnosis and forensic HLA typing.
Detection and isolation of homologous, repeated and amplified nucleic acid sequences is disclosed in U.S. Pat. No. 4,675,283. European Patent Application No. 0200362 describes a process for amplifying, detecting or cloning nucleic acid sequences and useful in disease diagnosis and in preparation of transformation vectors. A method for simple analysis of relative nucleic acid levels in multiple small samples by cytoplasmic dot hybridization is described in U.S. Pat. No. 4,677,054. A hybridization method of detecting nucleic acid sequences with a probe containing a thionucleotide is described in U.S. Pat. No. 4,647,529.
A simple and efficient enzymatic method for covalent attachment of DNA to cellulose and its application for hybridization-restriction analysis and for in vitro synthesis of DNA probes is described in Nucleic Acids Research (1986) 14: 9171-9191. Cleavage of single stranded oligonucleotides by Eco RI restriction endonuclease is described in Nucleic Acids Research (1987) 15: 709-716.
Exponential Amplification of Recombinant-RNA Hybridization Probes is described by Lizardi, et al. (1988) Bio/Technology 6:1197-1202. Fahrlander, et al., discusses Amplifying DNA Probe Signals: A Christmas Tree Approach in Bio/Technology (1988) 6:1165-1168. A nucleic acid hybridization assay employing probes cross-linkable to target sequences is described in U.S. Pat. No. 4,599,303.
A hybridization method and probe for detecting nucleic acid sequences is described in U.S. Pat. No. 4,908,307. An amplified hybridization assay is described in U.S. Pat. No. 4,882,269 wherein a family of signal-generating secondary probes bind to a primary probe that hybridizes to the target sequence of interest.
Detection of target sequences in nucleic acids by hybridization using diagnostic and contiguous probes for diagnosis of genetic abnormality diseases, especially in an automated procedure, is described in European Patent Application No. 0 185 494A2.
International Patent Application No. PCT/US89/02646 describes DNA amplification and subtraction techniques. Timblin, et al., discuss the application of PCR technology to subtractive DNA cloning and the identification of genes expressed specifically in murine plasmacytoma cells in Nucleic Acids Research (1990) 18(No.6): 1587-1593. Nelson, et al., disclose Alu PCR as a method for rapid isolation of human-specific sequences from complex DNA sources in Proc. Natl. Acad. Sci. USA (1989) 86: 6686-6690. The isolation of region-specific probes by Alu-PCR and coincidence cloning is discussed by de Jong, et al., in a publication of Lawrence Livermore Labs (1990).